Acylated 1,6-anhydro decasaccharide and its use as an antithrombotic agent

ABSTRACT

The instant invention relates to the decasaccharide of formula (I), wherein Ac represents an acetyl group: 
                         
in its acid form or in the form of any one of its pharmaceutically acceptable salts, and to its process of preparation. The oligosaccharide of formula (I) is useful as an antithrombotic agent.

The instant invention relates to a novel oligosaccharide, morespecifically an acylated 1,6-anhydro decasaccharide, and to its use asan antithrombotic agent.

Clotting is a defense mechanism preventing excessive loss of blood andingestion of microbes. Yet, inadvertent formation and dislocation ofclots may be harmful; antithrombotic drugs prevent the formation andgrowth of clots.

Heparin and Low Molecular Weight Heparins (LMWHs) are the currentstandard therapy in the management of thromboembolic diseases. Theiranticoagulant activity is exerted through inhibition of coagulationfactors, mainly activated factor X (FXa) and thrombin (factor IIa). Thisinhibitory action is mediated by the specific interaction of heparinspecies with antithrombin (AT), a serine protease inhibitor of theserpin family.

These drugs derive from animal sources: unfractionated heparin (UFH) isisolated from tissues such as lungs or intestinal mucosa, from porcineor bovine origins. LMWHs, such as tinzaparin, ardeparin, dalteparin,enoxaparin, nadroparin or reviparin, are obtained by enzymatic orchemical depolymerization of heparin.

Heparin and LMWHs are complex mixtures of molecules: they containnumerous sulfated polysaccharides, each of them being a polymer composedof a linear chain of monosaccharide residues. Therefore, the differentpolysaccharides present in heparin and in LMWHs vary in their lengths aswell as in their chemical structures. The varying degree of sulfationand the presence of different 1→4 linked uronic acid and glucosaminedisaccharide units give rise to a complex overall structure (J. Med.Chem., 2003, 46, 2551-2554).

Another class of antithrombotic drugs consists in syntheticoligosaccharides. Indeed, in the early 1980s it was determined that aunique pentasaccharide domain in some heparin chains is the minimalsequence required for binding and activating antithrombin III(Biochimie, 2003, 85, 83-89). Fondaparinux sodium is a syntheticanalogue of this pentasaccharide, obtained through more than 60 steps ofchemical synthesis. It is a selective inhibitor of factor Xa,commercialized for the prevention of thrombosis after orthopedic andabdominal surgery, for the prevention and treatment of deep veinthrombosis and pulmonary embolism, as well as for the treatment ofcoronary diseases.

Structure-based design has subsequently led to analogues with longerduration of action, such as idraparinux, displaying either selectivefactor Xa or dual Xa and IIa inhibition properties. The search forimproved pharmacodynamic profiles lead to the synthesis of longeroligosaccharides, such as the clinical candidate SR123781(hexadecasaccharidic compound), aiming at providing heparin mimeticsthat are more potent than heparin as regards antithrombin activity, butdevoid of its side effects.

The Applicant has devised a novel approach for the identification of newantithrombotic compounds. Starting from oligosaccharides mixtures ofLMWHs, specific analytical and separation methods have permitted toisolate an oligosaccharide endowed with advantageous antithromboticproperties, useful in anticoagulant therapy.

The oligosaccharide according to the instant invention responds to theformula (I), wherein Ac represents an acetyl group (i.e. a group offormula —COCH₃) and wherein the wavy line denotes a bond situated eitherbelow or above the plane of the pyranose ring:

The oligosaccharide of formula (I) is a decasaccharide. The inventionencompasses the decasaccharide of formula (I) in its acid form or in theform of any one of its pharmaceutically acceptable salts. In the acidform, the carboxylate (—COO⁻) and sulfate (—SO₃ ⁻) functional groups arerespectively in the —COOH and —SO₃H forms.

The term “pharmaceutically acceptable salt” of the oligosaccharide offormula (I) is understood to mean an oligosaccharide in which one ormore of the —COO⁻ and/or —SO₃ ⁻ functional groups are bonded ionicallyto a pharmaceutically acceptable cation. The preferred salts accordingto the invention are those for which the cation is chosen from thecations of alkali metals and more preferably still those for which thecation is sodium (Na⁺).

In accordance with the present invention, the compound of formula (I)can be obtained from a LMWH product by using orthogonal (combined)separation methods selected from Gel Permeation Chromatography (GPC), ATaffinity chromatography and High Performance Liquid Chromatography(HPLC), including dynamically coated anion exchange chromatography andcovalent anion exchange chromatography. According to the invention,these separation methods may be used in any possible combinationthereof.

Gel Permeation Chromatography can be performed on columns filled withBio Gel P30 (Bio-Rad) circulated with NaClO₄. Selected fractions aredesalted, using techniques known in the Art.

AT affinity chromatography can be performed on columns filled withAT-Sepharose. The stationary phase is prepared by coupling human AT (1g; Biomed) to CNBr-activated Sepharose 4B (Sigma). The methodology ofHöök et al. (FEBS Letters, 1976, 66(1), 90-3) is used to prepare the ATcolumn, which is eluted using a NaCl gradient.

Dynamically coated anion exchange chromatography HPLC is achieved usingCTA-SAX chromatography (dynamic anion exchange chromatography withcetyltrimethylammonium). CTA-SAX semi-preparative columns are coated asdescribed by Mourier, P. A. J. and Viskov, C. (Analytical Biochem.,2004, 332, 299-313) on columns filled with Hypersil BDS C18 (5 μm).Column coating is performed as for the analytical columns, bypercolating cetyltrimethylammonium hydrogen sulfate solutions inwater/methanol. Mobile phases are aqueous sodium methanesulfonate atconcentrations varying between 0 and 2.5 M. The pH is adjusted to 2.5 byaddition of diluted methanesulfonic acid. Collected fractions areneutralized and desalted on Sephadex G-10 after a preliminary treatmenton Mega Bondelut C18 cartridges (Varian).

Covalent anion exchange chromatography can be achieved using anionexchange on AS11 (Dionex) semi-preparative HPLC columns. Any other anionexchange method may be performed, using other columns than Dionex AS11.

A final step for desalting the oligosaccharide thus obtained isperformed, after neutralization of the collected fractions, in order torecover the oligosaccharide of the invention with the desired salt form.Methods for desalting oligosaccharides are well known to one of skill inthe Art; mention may be made for example of desalting on a Sephadex G-10column.

The following protocols describe in detail an example for thepreparation of the compound (I) according to the invention, in the formof a sodium salt. They are included herewith for purposes ofillustration only and are not intended to be limiting of the invention.

In this example, the compound (I) is prepared from a starting LMWHproduct by performing the following steps: Gel Permeation Chromatography(GPC), then ATIII affinity chromatography, then CTA-SAX chromatography(dynamically coated anion exchange chromatography), and then covalentanion exchange chromatography.

About 140 g of enoxaparin (commercially available from sanofi-aventis)are injected in about 60 runs in gel permeation, on columns (200 cm×5cm) filled with Bio Gel P30 and circulated with NaClO₄ 0.2 M at 100ml/h. Each run lasts about 24 hours. The decasaccharide fraction isgathered and desalted on a column filled with Sephadex G-10 (100 cm×7cm), circulated with water, to obtain about 18 g of said fraction.

The entire decasaccharide fraction is injected in ATIII affinitychromatography in about 36 runs where about 500 mg are injected on 30cm×5 cm columns. The low-affinity portion is eluted from the column witha 0.25 M NaCl solution buffered at pH 7.4 with 1 mM Tris at 6 ml/min.The high-affinity decasaccharide fraction is eluted with a step gradientof NaCl (0.7, 1.15, 1.6, 2.05, 2.65 and 3 M NaCl in 1 mM Tris-HCl, pH7.4). The NaCl gradient is monitored by the conductivity and thedetection is in UV at 232 nm.

Decasaccharides eluted in affine fractions with conductivities between30 and 85 mS/cm are gathered, desalted on Sephadex G-10, and used asstarting material for the next purification, achieved in CTA-SAX semipreparative chromatography (250 mm×22 mm columns). Column coating isperformed by percolating 1 mM cetyltrimethylammonium hydrogen sulfatesolutions in water/methanol (17:8, v/v) for 4 h with the columntemperature adjusted to 45° C. Mobile phases are aqueous sodiummethanesulfonate (Interchim) at concentrations varying between 0 and 2.5M. The pH is adjusted to 2.5 by addition of diluted methanesulfonicacid. Separations are achieved at 40° C. Salt concentration in themobile phase is increased linearly from 0 to 2.5 M over 60 min. Flowrate is 20 ml/min and UV detection at 234 nm is used.

About 800 mg are injected in 8 separate runs where 100 mg of the affinefraction are injected on the column. Fractions obtained are controlledon CTA-SAX analytical columns (150×2.1 mm Hypersil BDS C18 (3 μm)) afterneutralization. Fractions are gathered, passed through Mega Bondelut C18cartridges (Varian) and desalted on Sephadex G-10.

After that step, the decasaccharide of formula (I) is obtained withinsufficient purity, mainly due to its coelution with anotherdecasaccharide of formula (II).

The final purification is achieved on AS11 (Dionex) semi preparativecolumns, circulated with a NaClO₄ concentration gradient, for examplewith the following conditions:

Mobile phase: Solvent A: NaH₂PO₄, 2.5 mM, brought to pH 2.9 by addingH₃PO₄.

Solvent B: NaClO₄ in 1 N NaH₂PO₄, 2.5 mM, brought to pH 3.0 by addingH₃PO₄.

The elution gradient may be the following: T=0 min: % B=1; T=60 min: %B=80 and flow rate set at 20 ml/min. Detection is achieved in UV at 232nm.

Fractions are controlled on Dionex AS11 analytical columns (250×2.1 mm)and desalted on Sephadex G-10.

The decasaccharide (I) is obtained as a 50/50 mixture of glucosamine andmannosamine epimers (epimers at position 2 of the saccharide unit at thereducing end of the oligosaccharide, as noted by the wavy line). Theirproton NMR analyses, performed on a BRUCKER apparatus (600 MHz), are asfollows.

-   -   Glucosamine isomer:

NMR ¹H in D₂O (δ in ppm): 2.05 (3H, s), 3.18 (1H, s), 3.23 (2H, m), 3.32(1H, t, 6 Hz), 3.41 (1 H, dd, 8 et 2 Hz), entre 3.55 et 4.50 (38H, m),entre 4.55 et 4.62 (3H, m), 4.78 (3H, m), 4.95 (1H, s), 5.18 (1H, s),5.30 (1H, d, 3 Hz), 5.33 (3H, m), 5.45 (2H, m), 5.57 (1 H, s), 5.94 (1H, d, 6 Hz).

-   -   Mannosamine isomer:

NMR ¹H in D₂O (δ in ppm): 2.05 (3H, s), 3.23 (2H, m), 3.32 (1H, t, 6Hz), 3.42 (2H, m), entre 3.55 et 4.50 (38H, m), entre 4.55 et 4.62 (3H,m), 4.78 (3H, m), 4.95 (1H, s), 5.18 (1H, s), 5.30 (1H, d, 3 Hz), 5.33(3H, m), 5.45 (2H, m), 5.51 (1H, s), 5.94 (1 H, d, 6 Hz).

The oligosaccharide of the invention underwent pharmacological studieswhich demonstrated its antithrombotic properties and its value astherapeutically active substance.

Anti-FXa Activity in Plasma:

The ability of the sodium salt of the oligosaccharide (I) to accelerateAT-mediated FXa inhibition was analyzed in nearly physiologicalconditions. The anti-FXa activity measurement was performed using thecompetitive chromogenic assay STA®-Rotachrom® Heparin (Diagnostica StagoInc.) automated on a STA®-R analyzer (Diagnostica Stago Inc.) accordingto the manufacturer's recommendation. Bovine FXa (Diagnostica StagoInc.) was used. Fondaparinux was the reference material, obtained fromcommercial source marketed by GlaxoSmithKline. It was spiked atincreasing concentrations (0.0218-0.0460-0.0872-0.1740-0.3490-0.4650μmol/L) in normal pool human plasma (Hyphen). Dose response linearitywas demonstrated. The oligosaccharide of the invention and fondaparinuxwere tested at 6 concentrations ranging from 0.0218 to 0.4650 μM. Theconcentration of AT in plasma milieu was 2.25 μM. The measured absoluteanti-Xa activity of the purified oligosaccharide was expressed in IU/ml,according to European Pharmacopeia 6.0 (01/2008:0828). The relativeanti-FXa activity was calculated from the ratio of the absolute activityversus that of fondaparinux.

In this test, the oligosaccharide of the invention displays an absoluteanti-FXa activity of 1.21 IU/ml. Its relative anti-Xa activity comparedto fondaparinux is 1.49 fold.

The oligosaccharide of formula (I) according to the invention thereforedisplays high antithrombotic properties. It can be useful for thepreparation of drugs, specifically of antithrombotic drugs. Therefore,another object of the invention is a medicament, which comprises anoligosaccharide of formula (I) or an addition salt thereof with apharmaceutically acceptable salt.

Such a medicament is useful in therapeutics, in particular in thetreatment and prevention of thromboses, including venous thromboses (forexample in the post-operative phase of surgical patients, in cancerpatients or in medical patients with restricted mobility) and acutearterial thrombotic events, in particular in the case of myocardialinfarction.

Another object of the invention is also a pharmaceutical composition,which comprises, as active principle, an oligosaccharide of formula (I)according to the present invention. Such a pharmaceutical compositioncomprises an effective dose of an oligosaccharide of formula (I)according to the invention, or an addition salt thereof with apharmaceutically acceptable salt, and at least one pharmaceuticallyacceptable excipient. Said excipients are chosen according to thepharmaceutical form and the administration route desired, among usualexcipients known to one of skill in the art.

The pharmaceutical compositions according to the invention may comprise,in addition to the oligosaccharide of formula (I), at least one otheractive principle selected from antithrombotic oligosaccharides, whethersynthetic compounds (obtained by chemical, stepwise synthesis startingfrom appropriate mono- or oligosaccharidic building blocks) or compoundsisolated from heparin or LMWHs sources.

In the pharmaceutical compositions according to the invention for theoral, sublingual, sub-cutaneous, intramuscular, intra-venous, topical,local, intratracheal, intranasal, transdermal or rectal administration,the active principle of formula (I) above, or its salt, can beadministered as a unitary dosage form, in blend with usualpharmaceutical excipients, to animals and human beings for theprevention or for the treatment of the pathologies mentioned above.

The appropriate unitary dosage forms comprise the oral forms, such astablets, hard or soft gelatin capsules, powders, granules and oralsolutions or suspensions, the sublingual, buccal, intratracheal,intraocular, intranasal forms, by inhalation, the topical, transdermal,sub-cutaneous, intramuscular or intra-venous forms, the rectal forms andthe implants. For the topical application, the compound of the inventionmay be used as creams, gels, ointments or lotions.

The present invention, according to another of its aspects, also relatesto a method for the treatment and prevention of the above pathologies,which comprises the administration to a patient of an effective dose ofthe oligosaccharide of formula (I) according to the invention, or a saltwith a pharmaceutically acceptable salt thereof.

What is claimed is:
 1. An isolated and purified oligosaccharide offormula (I):

wherein Ac represents an acetyl group and wherein the wavy line denotesa bond situated either below or above the plane of the pyranose ring, inits acid form or in the form of any one of its pharmaceuticallyacceptable salts.
 2. The oligosaccharide according to claim 1, in theform of its sodium salt.
 3. A process for the preparation of anoligosaccharide according to claim 1, which comprises steps forseparating said oligosaccharide from a starting low molecular weightheparin (LMWH) by performing Gel Permeation Chromatography (GPC), ATaffinity chromatography, dynamically coated anion exchangechromatography (CTA-SAX) and covalent anion exchange chromatography, inany possible combination of those methods.
 4. The process according toclaim 3, which comprises the following steps: a) Gel PermeationChromatography (GPC), then b) AT affinity chromatography, then c)dynamically coated anion exchange chromatography (CTA-SAX), and then d)covalent anion exchange chromatography.
 5. The process according toclaim 3, wherein the anion exchange chromatography is performed onDionex AS11 HPLC columns.
 6. The process according to claim 3, whereinthe starting low molecular weight heparin (LMWH) is enoxaparin.
 7. Apharmaceutical composition, comprising an oligosaccharide of formula (I)according to claim 1, or a pharmaceutically acceptable addition saltthereof, and at least one pharmaceutically acceptable excipient.
 8. Thepharmaceutical composition according to claim 7, further comprising atleast one other active principle selected from antithromboticoligosaccharides.
 9. A method for the treatment of thromboses in apatient comprising administering to the patient an oligosaccharide offormula (I) according to claim 1, or a pharmaceutically acceptableaddition salt thereof.
 10. The method according to claim 9, wherein thethromboses are venous thromboses or acute thrombotic events.